Optical system and method for exciting and measuring fluorescence on or in samples treated with fluorescent pigments

ABSTRACT

An optical system and method of exciting and measuring fluorescence on or in samples treated using fluorescent pigments using such an optical system having at least one first laser ( 1 ); a mirror ( 4 ); a deflection element ( 7 ); an optic ( 8 ); and a unit ( 10 ), which includes mirror ( 4 ) and optic ( 8 ) mounted fixed in relation to one another. The unit ( 10 ) is positioned so it is linearly movable back and forth along the optical axis ( 5 ) and is mechanically connected to an oscillating linear drive ( 11 ). The optic ( 8 ) additionally acts as a collimator and the mirror ( 4 ) additionally acts to deflect the collimated light ( 12 ) parallel to the optical axis ( 5 ). The optical system additionally includes a table ( 13 ), movable at least in the direction of the X and Z spatial axes, for receiving sample holders ( 14 ) and for aligning the samples ( 6 ) in relation to a first focal point ( 9 ); an optical arrangement ( 16 ) for imaging a second focal point ( 17 ); an aperture plate ( 18 ) positioned in the second focal point ( 17 ); a first spectral filter ( 19 ); and a first detector ( 20 ) and is distinguished in that it includes a continuous geometrical axis (G), not running through the sample, on which the optical arrangement ( 16 ), the second focal point ( 17 ), the aperture plate ( 18 ), the first spectral filter ( 19 ), and the first detector ( 20 )—together with the mirror ( 4 ) and the deflection element ( 7 )—are positioned, this geometrical axis (G) being at least partially identical to the optical axis ( 5 ) in the region between the mirror ( 4 ) and the first detector ( 20 ).

[0001] The present intention relates to an optical system for excitingand measuring fluorescence on or in samples treated with fluorescentpigments according to the preamble of independent claim 1. Such opticalsystems are known, for example, as scanning light microscopes.

[0002] Scanning light microscopes have been known for several decades.Their functional principal is based on a light beam being concentratedto a small point of light (the first focal point) on a sample. Thesample and this point of light are mutually moved in such a way that aspecific area of the sample is scanned (rasterized) by the point oflight. The light which penetrates the sample or is reflected by itand/or the fluorescence triggered on or in the sample during thescanning is therefore referred to as “light originating from the sample”and is measured by one or more photodetectors. An enlarged image isproduced in that an original measurement point is assigned a specificarea on an image of the sample. In principle, such a scanning lightmicroscope therefore includes:

[0003] a light source, which produces a light beam;

[0004] a sample holder for holding the sample;

[0005] an optic for producing a first focal point on the sample;

[0006] an optical arrangement for imaging a second focal point using thelight which shines through the sample and/or is reflected by the sampleand/or which represents fluorescence triggered on or in the sample;

[0007] a photodetector for measuring the intensity of the second focalpoint; and

[0008] a scanning mechanism for mutual movement of the sample and firstfocal point.

[0009] In a conventional scanning light microscope, the light beam isdeflected in the direction of the two spatial axes X and Y to illuminatethe sample. This procedure hides the disadvantage that the angle ofincidence on the sample of the light refracted by the projective lensvaries and produces an aberration in the imaging of the sample light bythe objective lens. This aberration may be corrected through anappropriate construction of the objective lens. Such a lensdisadvantageously makes the optic more costly and simultaneously has alimiting effect in regard to the light collecting efficiency andoperating distance.

[0010] According to U.S. Pat. No. 5,081,350, this problem is solved inthat a device is disclosed therein using which the sample is scanned bya light beam. In this case, the device for illuminating the sample andthe device for measuring the signal coming from the sample are mountedon a unit which is movable back and forth. The sample is mounted on asample table movable perpendicularly to this oscillation in this case,so that scanning of the sample is possible with a constant angle ofincidence of the illumination. Because, especially for the applicationof a rapid scanning method, the light source is preferably to bepositioned outside the movable part of the scanning light microscope, inthis case the use of glass fiber waveguides is suggested, whichoptically connect the light source to the projective. However, there isthe danger that this glass fiber cable may be damaged by the frequentand rapid back and forth movement.

[0011] An improved device according to the species is known from U.S.Pat. No. 5,260,569, which solves the problems of the related artdescribed above in that a scanning light microscope is suggested thereinwhich, as a light source, includes a laser, a mirror for deflecting thelight, coming out of the laser and incident parallel to an optical axison the mirror, in the direction of a sample, a deflection element fordeflecting this light onto this mirror, an optic for producing a firstfocal point, a unit, including the mirror and the optic, in which themirror and optic are positioned fixed in relation to one another, whichis linearly movable back and forth along the optical axis, anoscillating linear drive which is mechanically connected to this unit, asample table, movable at least in the direction of the X and Z spatialaxes, for aligning the sample in relation to the first focal point, anoptical arrangement for imaging a second focal point using the lightoriginating from the sample, an aperture plate, positioned in the secondfocal point, for masking light originating from the sample which meetsthis aperture plate at a distance greater than a specific distance, aspectral filter for selecting a component of the light originating fromthe sample which passes through the aperture plate, and a detector formeasuring the intensity of the light originating from the sample whichpasses through the aperture plate and is selected by the spectralfilter. The optic is additionally implemented as a collimator for thelight originating from the sample and the mirror is additionallyimplemented for deflecting this collimated light diametrically oppositeto the direction of incidence of the light from the laser and parallelto the optical axis.

[0012] In addition to all features of the preamble of claim 1, U.S. Pat.No. 5,260,569 also discloses a scanning light microscope in which thelight emitted by a light source is aligned in parallel using acollimating lens acting as a part of the projective. The collimatedlight propagates in the direction parallel to the scanning direction ofthe microscope. Therefore, the collimated light beam—independently ofthe actual position of the unit movable back and forth—is alwaysincident from the same direction on the mirror which is fixed in theunit movable back and forth. This has the consequence that the lightbeam is always reflected by the mirror onto the sample in the samedirection and in collimated form. This collimated light is, after a 90°reflection on the mirror, bundled into a first focal point using afurther projective lens which is also fixed in the unit movable back andforth. Therefore, the scanning or rasterizing of the sample may beperformed using the unit movable back and forth and using the light of alight source which is attached to the unit movable back and forth.However, the attachment of the light source and photodetector outsidethe unit movable back and forth is preferable, so that this unit may bemade simpler and lighter—to allow more rapid scanning.

[0013] The object of the present invention is to suggest an alternativeoptical system and/or an alternative optical method which opens upadditional possibilities for a simpler, more flexible systemconstruction and/or system use and essentially has the advantages of therelated art.

[0014] According to a first aspect, this object is achieved by a systemcorresponding to the combination of features of independent claim 1, inwhich, in addition to the features of the preamble known from the mostsimilar related or closest prior art, it is suggested that the opticalsystem include a continuous geometrical axis G, which does not runthrough the sample, on which the optical arrangement, the second focalpoint, the aperture plate, the spectral filter, and thedetector—together with the mirror and the deflection element—arepositioned, this geometrical axis G being at least partially identicalto the optical axis in the region between the mirror and the detector.Further features according to the present invention result from thedependent claims.

[0015] According to a second aspect, this object is achieved by a methodcorresponding to the combination of features of independent claim 25, inwhich, in addition to the features of the preamble known from the mostsimilar related or closest prior art, it is suggested that an opticalsystem be provided having a continuous geometrical axis G which does notrun through the sample, on which the optical arrangement, the secondfocal point, the aperture plate, the spectral filter, and thedetector—together with the mirror and the deflection element—arepositioned, this geometrical axis G being at least partially identicalto the optical axis in the region between the mirror and the detector.Further features according to the present invention result from thedependent claims.

[0016] The present invention will now be described in greater detail onthe basis of schematic illustrations, which merely represent preferredexamples of embodiments and do not restrict the scope of the disclosurein regard to the present invention.

[0017]FIG. 1 shows a quasi-spatial schematic illustration of an opticalsystem according to a first embodiment having two lasers;

[0018]FIG. 2 shows a vertical partial section of an optical systemaccording to a second embodiment having at least one laser;

[0019]FIG. 3 shows a vertical partial section of an optical systemaccording to a third embodiment having at least one laser.

[0020]FIG. 1 shows a quasi-spatial schematic illustration of an opticalsystem of the reflection type according to a first embodiment. Thisoptical system is preferably capable of exciting and measuringfluorescence on or in samples treated using fluorescent pigments andincludes a first laser 1. This laser emits light of a first wavelength2—to excite first fluorescent pigments—so that these first fluorescentpigments emit light of a second wavelength 3. A mirror 4 deflects thelight of the first wavelength 2, which comes out of the first laser 1and is incident on the mirror 4 parallel to an optical axis 5, in thedirection of a sample 6 (cf. FIGS. 2 and 3). A deflection element 7deflects the light 2 from the first laser 1 onto this mirror 4. An optic8 produces a first focal point 9 for the incident light from the firstlaser 1 deflected by the mirror 4 of the first wavelength 2. A unit 10includes the mirror 4 and the optic 8. This mirror 4 and the optic 8 arepositioned fixed in relation to one another in the unit 10.

[0021] This unit 10 is positioned so it is movable back and forthlinearly along the optical axis 5 and is mechanically connected to anoscillating linear drive 11. The oscillating linear drive 11 may beimplemented, for example, as a stack of piezoelements; as a mechanicaldrive, including a connecting rod, for example, or as a naturallyoscillating solid body excited by ultrasound waves. However, theimplementation of an oscillating linear drive 11 as a “voice coil”, asis described in U.S. Pat. No. 5,260,569 and particularly in U.S. Pat.No. 5,880,465, is preferred. In this case, a device is used to producethe back and forth movement which essentially corresponds to aloudspeaker, the membrane movements being transferred to the unit 10using a mechanical connection.

[0022] This optic 8 is implemented accordingly, so that it additionallyacts as a collimator for the light of second wavelength 3 emitted by thefirst fluorescent pigments (“light originating from the sample”). Themirror 4 additionally deflects this collimated light 12 diametricallyopposite to the direction of incidence of the light of first wavelength3 and parallel to the optical axis 5. This optical system additionallyincludes a table 13, movable at least in the direction of the X and Zspatial axes, for samples 6 treated using at least one first fluorescentpigment and for aligning the sample 6 in relation to the first focalpoint 9. The table 13 is preferably additionally movable in thedirection of the Y spatial axis, the X axis and the Y axis running atleast approximately parallel to the surface 15 of a sample holder 14.

[0023] The X, Y, and Z spatial axes are at least approximatelyperpendicular to one another, the Y axis extending parallel to ahorizontally running part of the optical axis 5 and to the geometricalaxis G, which also runs horizontally.

[0024] The table 13 is preferably constructed in multiple parts andincludes a part 13′, movable in the direction of the X axis over asmaller distance (thin arrow), a part 13″, movable in the direction ofthe X axis over a larger distance (thicker double arrow), and a part13′″, movable in the direction of the Y axis over a larger distance(thicker double arrow). The entire table 13 or at least the part 13′ ismovable in the direction of the Z axis. Electric motors are preferablyused for moving the parts of the table 13.

[0025] Slides made of glass, for example, as have been known from lightmicroscopy for a long time, are suitable as sample holders 14. Otherand/or similar sample holders may be produced from plastic. Still other,essentially flat sample holders, which are also suitable for scanningtunnelling microscopy, for example, may be implemented from silicon orpyrolytic graphite and the like and/or include these materials. Sampleholders may also be used which have a defined partitioning on thesurface. This partitioning may include a uniform, grooved division;however, it may also have an array of depressions. Examples of sampleholders having such an array of depressions are silicon or glass plateshaving multiple etched depressions. Further examples are standardmicrotitration plates (trademark of Beckman Coulter, Inc., 4300 N.Harbour Blvd., P.O. Box 3100 Fullerton, Calif., USA 92834) or“microplates”, which include 96, 384, 1536, or more depressions in theform of “wells”.

[0026] While dry or at least partially dried or immobilized samples arepreferably prepared for investigation on slides, sample holder 14 havingdepressions and/or wells may accommodate liquid samples 6 or samples 6located in a liquid. A frame 31 which essentially has the externaldimensions of a microplate has been shown to be especially suitable foruse in the optical system according to the present invention. This frametherefore fits in and/or on the same sample table 13 as a microplate andmay also be placed there or removed therefrom automatically like amicroplate, i.e., using a robot arm. This frame 31 is implemented forreceiving and holding multiple slides, particularly made of plastic,glass, silicon, pyrolytic graphite, and the like. The arrangement of 4glass slides on such a frame has especially proven itself. In this case,the object carriers 14 are arranged, as shown in FIG. 1, for example, ina row, long edge to long edge—each pushed together or at a slightdistance to one another (cf. FIG. 1). The frame 31 is preferablyproduced from plastic in the injection molding method, through which theproduction price may be kept low and the dimensional precision may bekept high. This dimensional precision makes using such frames 31 in allpossible devices for automated handling of microplates significantlyeasier. Of course, the frames 31 may also be equipped with the slides 14and inserted into the optical system according to the present inventionby hand. Frames 31 equipped with slides 14 additionally ease the furtherhandling of slides secured in this way, which now no longer have to begrasped directly between the process steps to be performed in thedevices for automated handling.

[0027] The optical system additionally includes an optical arrangement16 for imaging a second focal point 17 using the light of the secondwavelength 3 emitted by the first fluorescent pigments, collimated bythe optic 8, and deflected by the mirror 4 (“light originating from thesample”). An aperture plate 18 positioned in the second focal point 17is used for masking light of the second wavelength 3 which meets thisaperture plate 18 at a distance greater than a specific distance fromthe focal point 17. This aperture plate 18 is preferably implemented asreplaceable, so that using multiple diameters of this confocal apertureplate, the sharpness and/or the brightness of the second focal point maybe increased or reduced as required. Especially in the event ofextremely poor light conditions, a larger plate, which causes areduction of the imaging sharpness, may be useful. A replaceableaperture plate 18 allows selection of different aperture diameters foradjusting the imaging in the second focal point 17 to the sample volumeand/or to the desired penetration depth of the excitation beam into thesample and selection of the corresponding depth of field of the detectorarrangement.

[0028] The optical system also includes a first spectral filter 19 forselecting a component of the light of the second wavelength 3 passingthrough the aperture plate 18 and a first detector 20 for measuring theintensity of the light of the second wavelength 3 transmitted throughthe aperture plate 18 and selected by the first spectral filter 19(“light originating from the sample”).

[0029] The optical system according to the present invention includes acontinuous geometrical axis G, which does not run through the sample, onwhich the optical arrangement 16, the second focal point 17, theaperture plate 18, the first spectral filter 19, and the first detector20 are positioned—together with the mirror 4 and the deflection element7—this geometrical axis G being at least partially identical to theoptical axis 5 in the region between the mirror 4 and the first detector20.

[0030] This joint arrangement on the geometrical axis G has theadvantage that the scanning of the sample may be performed at high speedusing a very simply constructed unit 10 movable back and forth. Thisunit 10 actually only includes the mirror 4 and the optic 8. Theoscillation frequency is preferably 20 Hz, the oscillation amplitude Dable to be up to 25 mm or more.

[0031] The light of the laser 1 placed distal from this unit 10 and alsothe light, originating from the sample, running parallel anddiametrically opposite to that of the laser 1, represents a purelyoptical connection between the scanning part of the system and theexcitation and measurement part of the system, so that no oscillationsare transferred from the scanning or raster part to the measurement partof the system. In addition, this arrangement allows the bundled light 2of the laser 1 to be fed directly into the optical system. The mirror 4deflects the bundled laser beam 2 onto the optic 8, which in turn alwaysdeflects the resulting focal point onto the same point on the opticalaxis 5. This optical axis 5 preferably runs vertically between themirror 4 and the sample 6. This optical system, based on coupling abundled laser beam, is subject to fewer light losses than those whichfeed and deflect a collimated light beam in order to obtain a firstfocal point.

[0032] In the system according to the present invention, the opticalaxis 5 is identical to the geometrical axis G along a part whichpreferably runs horizontally. In a direction parallel to these axes 5,G,a bundled laser beam is used to excite the sample, and a collimated beamhaving light originating from the sample is used in the directiondiametrically opposite thereto. In this way, the deflection element 7,which is to diffract the laser beam 2 in the direction of the mirror 4,but which is to be transparent to the collimated light beam, may be asimple glass plate. Typically, approximately 4% of a light beam isreflected and approximately 96% is transmitted at a boundary surfaceglass/air. Antireflection coating increases the transmission ifnecessary and reduces the reflection of such a glass plate. This simpleglass plate or disk replaces the more expensive polarization beamsplitter known from U.S. Pat. No. 5,260,569 (see FIG. 1 therein,reference number 13) and quarter-wave plates (see FIG. 1 therein,reference number 18), which are necessary in the related art because acollimated light beam is used in both directions, each of which requiresthe entire surface of the mirror and filter.

[0033] A further advantage of the system according to the presentinvention is that the laser beam 2 for exciting the sample fluorescencemust run parallel to the optical axis 5, but not in its center and notin the center of the geometrical axis G. This is advantageous above allfor determining a mutually optimum Z position of sample 6 and firstfocal point 9. The laser beam then preferably runs at a distance (A) tothe optical axis 5, which is identical to the geometrical axis G.Therefore, “off-center illumination” of the sample is possible, as isshown in FIGS. 2 and 3, for example.

[0034] Furthermore, the system according to the present inventionincludes the possibility of using a preferred deflection element 7,which, for deflecting the light 2 from the first laser 1 onto thismirror 4 in a direction parallel to the optical axis 5, includes ahighly reflective region 21 intended for the laser beam 2 and isessentially transparent in its remaining regions to the light of boththe first wavelength 2 and the second wavelength 3. Therefore, if such a“pin mirror” is used, a greatly improved yield for the bundled laserlight for exciting the fluorescence of the fluorescent pigments in or onthe samples 6 results in comparison to the simple glass plate used asthe deflection element 7. The loss of approximately 5% of the lightoriginating from the sample and deflected on the highly reflectiveregion 21 is not of great importance here.

[0035] In order to make the device more compact, the optical system mayadditionally include a simple mirror for reflecting the light beamsrunning between the deflection element 7 and the mirror 4 (not shown).This simple mirror is then also positioned in the geometrical axis (G).However, in this case the optical arrangement 16, the second focal point17, the aperture plate 18, the first spectral filter 19, and the firstdetector 20—corresponding to the deflection by the simple mirror—arepositioned on a geometric axis G′, which is different from G.

[0036] The optical system shown in FIG. 1, having two preferablymonochromatic lasers of different wavelengths, additionally includes asecond laser 1′, which—to excite second fluorescent pigments—emits lightof a third wavelength 2′running parallel to the light of the firstwavelength 2, so that these fluorescent pigments emit light of a fourthwavelength 3′. A second spectral filter 19′ selects a component of thelight of the fourth wavelength 3′ and a second detector 20′ measures theintensity of the part of the light of the fourth wavelength 3′ selectedby the second spectral filter 19′. In addition, a beam splitter element26 is provided, which is at least partially transparent to the light 12of the second wavelength 3 emitted by the first fluorescent pigments andcollimated by the optic 8 and which is implemented for reflecting and/ordeflecting the light of the fourth wavelength 3′ from the geometric axisG in the direction of a second detector 20′. This beam splitter element26 may be designed as a dichroic mirror or even, for example, as a 50%beam splitter.

[0037] An object illuminator 30 in the form of a simple lamp, forexample, is preferably also provided distal from the unit 10, so thatthe objects to be scanned may be observed and/or imaged using lightoptics if necessary. The light used is fed using a dichroic mirrorpositioned on the axes 5, G. The spectral filters 19, 19′ are designedin such a way that they filter out this light. A separate detector (notshown) may be provided in order to capture the visual image of thesamples 6. Alternatively, the detector 20 may also be used in order toscan the samples in the fluorescence mode, for example.

[0038]FIG. 2 shows a vertical partial section of an optical system forexciting and measuring fluorescence on or in samples treated usingfluorescent pigments according to a second embodiment. This opticalsystem includes all features of claim 1. For this second, somewhatsimpler embodiment, only one monochromatic laser 1 is provided. Thetable 13 is additionally movable in the direction of the Y spatial axis,X and Y axes running at least approximately parallel to the surface 15of a sample holder 14. The sample holders described in FIG. 1 may allalso be used in this embodiment.

[0039] The deflection element 7, for deflecting the light 2 from thefirst laser 1 onto this mirror 4 in a direction parallel to the opticalaxis 5, includes a highly reflective region 21 intended for the laserbeam 2. In its remaining regions, the deflection element 7 isessentially transparent to the light of both the first wavelength 2 andthe second wavelength 3. The highly reflective region 21 of thedeflection element 7 is positioned at a distance A to the optical axis5, which is identical to the geometrical axis G, so that the beam pathfor the light of the first wavelength 2 coming out of the laser 1 runsparallel and at a distance A to these axes 5, G.

[0040] An opaque screen 22 is positioned between the deflection element7 and the optical arrangement 16 for intermittent masking of a laserbeam, used for focusing and excitation and reflected by the sample 6. Inthe state shown, this screen 22 is pushed into the beam path.

[0041] The bundled light 2 coming from the laser 1 is deflected on thehighly reflective region 21 of the deflection element 7 in the directionof the mirror 4—preferably slanted at 45° to the horizontal—parallel tothe axes 5, G and at a distance A to these axes. The mirror 4 deflectsthis bundled laser beam 2 parallel and at the distance A to the now atleast approximately vertically running optical axis 5, upon which thelaser beam is deflected into the focal point 9 lying in the optical axis5. The previously described course of the laser beam 2 is used forfocusing, i.e., for fixing an optimum operating distance between optic 8and sample 6. At the same time, the mutual position of focal point 9 andsample 6 is determined and set (as described below). The focusing beam25 concentrated on the focal point 9 has a spot diameter between thediffraction limit and 2 mm. A preferred spot diameter is less than 15μm. The focusing beam 25 is reflected by the sample and, at a distancefrom the optical axis 5, reaches the mirror 4 again, which deflects thisreflected beam in the direction parallel to the horizontally runningaxes 5, G.

[0042] This off-center illumination is especially suitable fordetermining an exact operating distance—which is preferably up to 7 mm,but may also be more or less—because the laser beam is incident on thesample at an angle less than 90°. It is obvious that as the angle ofincidence is reduced, the precision and/or the resolution in thedirection of the Z axis increases, while an angle of incidence of 90°allows the lowest resolution in the direction of the Z axis, becauseunder certain circumstances it may not be determined, for example, atwhat depth the focal point 9 is located inside the sample 6. In additionto this, the resolution of the optical system in the direction of the Zaxis increases with increasing distance A. The reflected beam runningparallel to the axes 5, G is deflected by an optical arrangement 16 intothe second focal point 17, where it passes through the aperture plate18. The spectral filter is now preferably pulled out of the beam path orreplaced by a neutral density filter (gray filter), so that thereflected beam is incident on the detector 20 and the detector maymeasure the intensity of the reflected beam.

[0043] If the optimum operating distance is not to be set using thereflection, but rather on the basis of the intensity of the fluorescenceexcited in the sample(s) and/or on the basis of diffuse scattered lightgenerated in or on the sample 6, the screen 22 is pushed into the beampath (cf. FIG. 2) and the spectral filter 19 and/or a neutral densityfilter (gray filter) is placed in front of the detector 20. The bundledlaser beam 1 is then used as the excitation beam 24, which is deflectedinto the first focal point 9 in the same way as the focusing beam 25. Apart of the fluorescence, which propagates essentially in a dome shapefrom the sample 6, is collimated (i.e., aligned in parallel) in theoptic 8 and deflected by the mirror 4 in the direction of the opticalarrangement 16. In spite of a small loss, which is essentiallydetermined by the area of the highly reflective region 21 of thedeflection element 7 and the screen 22, this collimated fluorescentlight is incident at the optical arrangement 16 and is focused in thesecond focal point 17. There—depending on the diameter of the apertureplate 18 selected—a certain part of the focused fluorescent light passesthrough this aperture plate 18 and the spectral filter 19 and isdetected by the detector 20. The same beam path as just described isused for scanning the sample.

[0044] The first spectral filter 19 is positioned between the apertureplate 18 and the first detector 20. It may also be used as a window inthe detector 20. However, the implementation of the first spectralfilter 19 as a filter slider having, for example, five different filterswhich may be automatically replaced with one another is preferred, thisspectral filter 19 being manually replaceable by another filter sliderhaving another filter set.

[0045] This optical system preferably includes a computer ormicroprocessor for recording and processing the measurement signalsdetected by the detector 20 and for outputting data corresponding tothese signals. This computer or microprocessor is preferably alsoimplemented for controlling the movements of the table 13. The table 13,which is displaceable in the directions of the X and/or Y and/or Z axes,is preferably also implemented so it may be tilted around the X and/or Yaxes.

[0046] According to the method according to the present invention, suchan optical system is provided with a continuous geometrical axis G,which does not run through the sample. The optical arrangement 16, thesecond focal point 17, the aperture plate 18, the first spectral filter19, and the first detector 20—together with the mirror 4 and thedeflection element 7—are positioned on this geometrical axis G. In thiscase, this geometrical axis G is at least partially identical to theoptical axis 5 in the region between the mirror 4 and the first detector20.

[0047] During the excitation and measurement of the fluorescence emittedby the sample 6, the unit 10 of this optical system is fixed accordingto a first type of use. The sample table 13 is simultaneously moved inthe direction of the X and/or Y axes, the Y axis running parallel to theaxes 5, G. Using this table movement, which may be performed by theparts 13′ (X movement) and 13′″ (Y movement), for example, a linear scanon or in the samples 6 is made possible with continuous excitation andmeasurement, and a point scan is made possible with point excitation andpoint measurement.

[0048] During the excitation and measurement of the fluorescence emittedby the sample 6, the unit 10 of this optical system is moved back andforth in the direction of the Y axis according to a second type of use.The sample table 13 is simultaneously fixed and/or moved in thedirection of the X axis. Using only these movements of the unit 10, alinear scan on or in the samples 6 is made possible with continuousexcitation and measurement, and with simultaneous movement of unit 10 (Ymovement) and table 13 (movement of the part 13′ results in X movement)an area scan is made possible with continuous excitation andmeasurement. The Y axis runs parallel to the axes 5, G in this case. Theoscillating linear drive 11 for the unit 10 is preferably implemented asa “voice coil”.

[0049] Before the effective scanning (measurement) of the samples, the Zposition,of the sample holder is set.

[0050] Laser pulses or lamp flashes may be used as an alternative lightsource for the excitation of the fluorescence. Discrete, individuallaser pulses or discrete short series of a few laser pulses are thenpreferred, which excite the fluorescence at one point at a time in or onsamples 6. Such point measurements are preferably performed especiallyduring measurement of the fluorescence in samples provided in the 1536wells of a high-density microplate, for example. Between or during themeasurements, unit 10 and sample holder 14 are displaced in relation toone another in this case, the displacement of the unit 10 in thedirection of the Y axis above all able to be performed extremelyrapidly. The measurement of the fluorescence in these 1536 wells, whichlasts approximately 1 minute in area scan with continuous excitation andmeasurement using the most rapid current devices, may thus be shortenedto approximately 10 seconds. For microplates having even more wells oran even smaller scanning dimension, an even larger time savings results.

[0051] For samples 6 immobilized on essentially flat sample holders 14,for example, the opaque screen 22, which is positioned between thedeflection element 7 and the optical arrangement 16, is pulled out ofthe beam path to define an optimum Z position of the movable table 13and/or a sample 6. Subsequently—on the basis of a series of measurementsignals generated during a Z movement of the table 13 by the firstdetector 20 and recorded in a computer or microprocessor—the Z positionof the table corresponding to the maximum of these measurement signalsis calculated and the table 13 is moved into this Z position. Thegeometric center between the points of inflection of the rising andfalling slopes of the measurement signal is preferably taken to fix themaximum of these measurement signals. This method is preferablyperformed at at least three points of the sample holder and the table 13is displaced—in accordance with the defined maxima for these threepoints—in the directions of the X, Y, and Z axes and tilted around the Xand Y axes as far as necessary. It is advantageous if this definition ofan optimum Z position is performed using a computer or microprocessor,which detects and processes the measurement signals generated by thedetector 20, outputs data corresponding to these signals, and alsocontrols the movements of the table 13.

[0052]FIG. 3 shows a vertical partial section of an optical system forexciting and measuring fluorescence on or in samples treated usingfluorescent pigments according to a third embodiment. This opticalsystem includes all features of claim 1. One or more preferablymonochromatic lasers 1, 1′ may be provided for this third embodiment.The table 13 is preferably movable in the direction of the X, Y, and Zspatial axes, X and Y axes running at least approximately parallel tothe surface 15 of a sample holder 14. In addition, the table 13 maypreferably be tilted around the X and/or Y axes. The sample holdersdescribed in FIGS. 1 and 2 may all also be used in this embodiment.

[0053] The deflection element 7 for deflecting the light 2 from thefirst laser 1 onto this mirror 4 in a direction parallel to the opticalaxis 5 includes a highly reflective region 21 intended for the laserbeam 2. In its remaining regions, the deflection element 7 isessentially transparent to the light of both the first wavelength 2 andthe second wavelength 3. The highly reflective region 21 of thedeflection element 7 is positioned in the center of the optical axis 5,which is identical to the geometrical axis G, so that the beam path forthe light of the first wavelength 2 coming out of the laser 1 runsparallel to and in the center of these axes 5, G. This optical systemincludes a separating element 23 for separating the light of the firstwavelength 2 coming out of the laser 1 into an excitation beam 24 forthe fluorescence and a focusing beam 25 parallel to this excitation beam24. An opaque screen 22 is positioned between the deflection element 7and the separating element 23 for intermittent masking of a laser beamused to excite the fluorescence. This separating element 23 may beimplemented as a simple, plane-parallel glass plate having completemirroring on the back, if only one laser 1 is to be used.

[0054] The bundled light 2 from the laser 1 is reflected approximately4% in the direction of the deflection element 7 on the non-mirroredfirst surface of the separating element 23. There, this laser beam isincident on a non-mirrored region and is again reflected approximately4% and at a distance A parallel to the axes 5, G in the direction of themirror 4. The bundled light 2 coming from the laser 1 is additionallyreflected on the rear surface of the separating element 23. With a glassplate completely mirrored on the back, approximately 96% of the laserlight 2 on optical axis 5 is deflected on the deflection element 7,where the light 2 is incident on its highly reflective region 21. Theseparating element 23 thus fulfills the object of separating the laserbeam 2 into an excitation beam 24 running on the optical axis 5 and afocusing beam 25 running parallel thereto at a distance A.

[0055] A simple, plane-parallel glass plate which is placed diagonallyin the beam path may also be used as an alternative separating element.The position of this plane-parallel glass plate is to be located betweenthe laser 1 and the deflection element 7; however, this plane-parallelglass plate is preferably positioned between the laser 1 and theseparating element 23 (not shown). The largest component of the laserbeam passes through the glass plate with a slight parallel offset. Inthis case, approximately 4% is reflected back into the glass plate atthe rear boundary surface glass/air. A component of approximately 4% ofthe reflected beam then subsequently experiences another reflection atthe front boundary surface. This leads to a small component of the laserlight being deflected parallel to the main beam in the direction of thedeflection element 7. In this case, the separating element 23 isimplemented as a front surface mirror and is not used to separate thebeam.

[0056] If two or more monochromatic lasers of different wavelengths areto be used, a plane-parallel plate implemented as a dichroic mirror isused as the separating element 23. Each laser may then—in accordancewith the wavelength of its light—be assigned its own separating element23, 23′, which is transparent to the remaining lasers. These separatingelements are then preferably positioned on a shared optical axis anddeflect the laser beams in the direction of one single deflectionelement 7, as may be seen from FIG. 1.

[0057] The bundled light 2, 2′ coming from the lasers 1, 1′ is deflectedon the highly reflective region 21 of the deflection element 7 in thedirection of the mirror 4 preferably slanted at 45° to the horizontal.

[0058] The stronger of the two beams produced by the separating element23 is therefore preferably incident on the highly reflective region 21of the deflection element 7, where it is deflected, in the direction ofthe optical axis 5 and the geometrical axis G, identical thereto, ontothe mirror 4. This excitation beam 24, which is preferably masked forfocusing using inserted screen 22, is deflected by mirror 4 in thedirection of the at least approximately vertically running optical axis5, passes through the optic 8, and is incident on the sample 6 in theoptical axis 5. The excitation beam 24, which is incident on the sample6 at least approximately vertically, is especially suitable for excitingthe fluorescence in samples 6 which are provided immersed in a liquid orin liquid form. Such samples are preferably provided in the wells ofmicroplates for investigation. Even if high-density microplates havingnot only 96, but 384, 1536, or more wells are to be used, the at leastapproximately vertical excitation beam always reaches the samples 6.

[0059] In contrast to the use of known and expensive, so-called“f(θ)-optics” for achieving an excitation beam which is incident on asample 6 at least nearly perpendicularly, the present invention allowsthe use of significantly more cost-effective optical elements for themirror 4 and the optic 8.

[0060] The second, preferably weaker light beam is thus incident on anon-mirrored region of the deflection element 7 where it is(approximately 4%) deflected, running parallel to the direction of theaxes 5, G and at the distance A thereto, onto the mirror 4. Thisfocusing beam 25 is preferably only active when screen 22 is insertedand is deflected by mirror 4 parallel and at a distance A to thedirection of the at least approximately vertically running optical axis5. The optic 8 deflects the focusing beam onto the focal point 9 lyingin the at least approximately vertical optical axis 5.

[0061] The considerations of the use of the focusing beam (cf. secondembodiment, FIG. 2) and of the focusing on the basis of the reflection,i.e., for fixing an optimum operating distance between optic 8 andsample 6, also essentially apply here. This is also true for focusingusing measurement of the fluorescence and/or on the basis of diffusescattered light generated in or on the sample 6, the screen 22 remainingpulled out of the beam path if the third embodiment described here isused and the fluorescence is measured.

[0062] A part of the fluorescence propagating essentially in a domeshape from the sample 6 is collimated (i.e., aligned in parallel) in theoptic 8 and deflected by the mirror 4 in the direction of the opticalarrangement 16. In spite of a small loss, which is essentiallydetermined by the area of the highly reflective region 21 of thedeflection element 7, this collimated fluorescent light is incident atthe optical arrangement 16 and is focused in the second focal point 17.There, a certain part of the focused fluorescent light—depending on theselected diameter of the aperture plate 18—passes through this apertureplate 18 and the spectral filter 19 and is detected by the detector 20.The same beam path as just described is used for scanning the sample.

[0063] The excitation beam, which always runs in the optical axis 5here, is reflected by the sample 6 and runs on the diametricallyopposite path in the direction of the lasers 1, 1′.

[0064] If two monochromatic lasers 1, 1′ are used, a beam splitterelement 26 is preferably positioned between the aperture plate 18 andthe first detector 20 (cf. FIG. 3). A first convergent lens 28 is usedfor collecting the light of the second wavelength 3 passing through theaperture plate 18. A second convergent lens 28′ is positioned downstreamfrom the beam splitter element 26, using which the light of the fourthwavelength 3′ passing through the aperture plate 18 is collected andsupplied to the second detector 20′ having the second spectral filter19′.

[0065] If necessary, further optical elements (lenses, mirrors, screens)may be placed between the aperture plate 18 and the detector20—preferably between the spectral filter 19 and the detector 20—foroptimized beam guiding in regard to signal yield and filter effect.

[0066] The reference numbers in the different figures each refer toidentical features. Any arbitrary combinations of the embodiment shownand/or described are included in the scope of the present invention.

[0067] Advantages which differentiate the present invention from therelated prior art include:

[0068] the use of sample holders 14 the size of a microplate or evenlarger sample holders is made possible;

[0069] the operating distance may be up to 7 mm or more;

[0070] the numerical aperture of the objective is above a value of 0.4and is up to 0.6 or more;

[0071] the oscillation amplitude D may be up to 25 mm or more, inparticular thanks to the use of a counter-oscillator 29;

[0072] the at least approximately vertical excitation of the samples isachieved using a cost-effective and largely aberration-free optic;

[0073] the deflection element 7 may be implemented as a simple glassdisk or as a pin mirror and allows the separation of excitation beam andfluorescence as a simple optical element;

[0074] the use of a deflection element 7 in the form of a pin mirrorallows the simultaneous use of multiple monochromatic lasers ofdiffering wavelengths and the measurement of a nearly unlimited numberof different fluorescent pigments of differing emission wavelengths;

[0075] the exchange of two monochromatic lasers of different wavelengthsdoes not require any further modification of the optical system;

[0076] the use of the different focusing modes disclosed in combinationwith an aperture plate of different aperture diameter allows greatlyvarying samples and sample formats to be brought into focus, excited,and their fluorescence measured.

1. An optical system, for exciting and measuring fluorescence on or insamples treated using fluorescent pigments, including: at least onefirst laser (1) which emits light of a first wavelength (2) to excitefirst fluorescent pigments, so that these first fluorescent pigmentsemit light of a second wavelength (3); a mirror (4), for deflecting thelight of the first wavelength (2), which comes out of the first laser(1) and is incident on the mirror (4) parallel to an optical axis (5),in the direction of a sample (6); a deflection element (7) fordeflecting the light (2) from the first laser (1) onto this mirror (4);an optic (8), for producing a first focal point (9) for the light of thefirst wavelength (2), which is incident from the first laser (1) and isdeflected by the mirror (4); a unit (10), which includes the mirror (4)and optic (8), the mirror and optic being positioned fixed in relationto one another in the unit and this unit (10) being positioned so it islinearly movable back and forth along the optical axis (5) and beingmechanically connected to an oscillating linear drive (11), the optic(8) additionally being implemented as a collimator for the light of thesecond wavelength (3) emitted by the first fluorescent pigment and themirror (4) additionally being implemented for deflecting this collimatedlight (12) diametrically opposite to the direction of incidence of thelight of the first wavelength (2) and parallel to the optical axis (5);a table (13), movable at least in the direction of the X and Z spatialaxes, for receiving sample holders (14) for samples (6) treated using atleast one first fluorescent pigment and for aligning the sample (6) inrelation to the first focal point (9); an optical arrangement (16) forimaging a second focal point (17) using the light of the secondwavelength (3), which is emitted by the first fluorescent pigment,collimated by the optic (8), and deflected by the mirror (4); anaperture plate (18), positioned in the second focal point (17), formasking light of the second wavelength (3), which is incident on thisaperture plate (18) at a distance from the focal point (17) greater thana specific distance; a first spectral filter (19) for selecting acomponent of the light of the second wavelength (3) passing through theaperture plate (18); and a first detector (20) for measuring theintensity of the light of the second wavelength (3) which passes throughthe aperture plate (18) and is selected by the first spectral filter(19), characterized in that the optical system includes a continuousgeometrical axis (G), which does not run through the sample, on whichthe optical arrangement (16), the second focal point (17), the apertureplate (18), the first spectral filter (19), and the first detector(20)—together with the mirror (4) and the deflection element (7)—arepositioned, this geometrical axis (G) being at least partially identicalto the optical axis (5) in the region between the mirror (4) and thefirst detector (20).
 2. The system according to claim 1, characterizedin that the table (13) is additionally movable in the direction of the Yspatial axis, X and Y axes lying at least approximately parallel to thesurface (15) of the sample holder (14).
 3. The system according to claim1 or 2, characterized in that it additionally includes a simple mirrorfor reflecting the light beams running between the deflection element(7) and the mirror (4), this simple mirror also being positioned in thegeometrical axis (G), the optical arrangement (16), the second focalpoint (17), the aperture plate (18), the first spectral filter (19), andthe first detector (20)—in accordance with the deflection by the simplemirror—being positioned on a geometrical axis (G′) different from (G).4. The system according to one or more of the preceding claims,characterized in that the deflection element (7) includes a highlyreflective region (21), intended for the laser beam (2), for deflectingthe light (2) from the first laser (1) onto this mirror (4) in adirection parallel to the optical axis (5), and in its remaining regionsis essentially transparent to the light of both the first wavelength (2)and the second wavelength (3).
 5. The system according to claim 4,characterized in that the highly reflective region (21) of thedeflection element (7) is positioned at a distance (A) to the opticalaxis (5), which is identical to the geometrical axis (G), so that thebeam path for the light of the first wavelength (2) coming out of thelaser (1) runs parallel and at the distance (A) to these axes (5, G). 6.The system according to claim 5, characterized in that an opaque screen(22)—for intermittent masking of a laser beam used for focusing andexcitation and reflected from the sample (6)—is positioned between thedeflection element (7) and the optical arrangement (16).
 7. The systemaccording to one or more of claims 1 through 4, characterized in that itadditionally includes a separating element (23) for separating the lightof the first wavelength (2) coming out of the laser (1) into anexcitation beam (24) and a focusing beam (25), which is parallel to thisexcitation beam (24).
 8. The system according to claim 7, characterizedin that an opaque screen (22)—for intermittent masking of a laser beamused for exciting the fluorescent pigments—is positioned between thedeflection element (7) and the separating element (23).
 9. The systemaccording to one or more of the preceding claims, characterized in thatthe first spectral filter (19) is positioned between the aperture plate(18) and the first detector (20).
 10. The system according to one ormore of the preceding claims, which includes: a second laser (1′),which—to excite second fluorescent pigments—emits light of a thirdwavelength (2′) running parallel to the light of the first wavelength(2), so that these fluorescent pigments emit light of a fourthwavelength (3′); a second spectral filter (19′) for selecting acomponent of the light of the fourth wavelength (3′); and a seconddetector (20′) for measuring the intensity of the part of the light ofthe fourth wavelength (3′) selected by the second spectral filter (19′),characterized in that it additionally includes a beam splitter element(26), which is at least partially transparent to the light (12) of thesecond wavelength (3) emitted by the first fluorescent pigments andcollimated by the optic (8), and which is implemented for reflectingand/or deflecting the light of the fourth wavelength (3′) from thegeometric axis (G) in the direction of a second detector (20′).
 11. Thesystem according to claim 1 or 10, characterized in that it additionallyincludes at least one convergent lens (28, 28′) for collecting the lightof the second wavelength (3) and/or fourth wavelength (3′) passingthrough the aperture plate (18).
 12. The system according to claim 10 or11, characterized in that the beam splitter element (26) is positionedbetween the aperture plate (18) and the detector (20).
 13. The systemaccording to one or more of the preceding claims, characterized in thatthe sample holder (14) which may be received by the sample table (13) isimplemented as a slide, particularly made of plastic, glass, silicon,pyrolytic graphite, and the like.
 14. The system according to one ormore of the preceding claims 1 through 12, characterized in that thesample holder (14) which may be received by the sample table (13) isimplemented as a microplate, particularly having 96, 384, 1536, or morewells.
 15. The system according to one or more of preceding claims 1through 12, characterized in that the sample holder (14) which may bereceived by the sample table (13) is implemented as a frame (31) forreceiving and holding multiple slides, particularly made of plastic,glass, silicon, pyrolytic graphite, and the like, and has the externaldimensions of a microplate.
 16. The system according to one or more ofthe preceding claims, characterized in that the unit (10)—during theexcitation and measurement of the fluorescence emitted by the samples(6)—may be fixed and the sample table (13) is simultaneously implementedas movable in the direction of the X and/or Y axes, the Y axis runningparallel to the axes (5, G).
 17. The system according to one or more ofpreceding claims 1 through 15, characterized in that the sample table(13)—for excitation and measurement of the fluorescence emitted by thesamples (6)—is implemented as movable in the direction of the X axis andthe unit (10) is simultaneously movable back and forth in the directionof the Y axis, the Y axis running parallel to the axes (5, G).
 18. Thesystem according to claim 17, characterized in that the oscillatinglinear drive (11) is implemented as a “voice coil”.
 19. The systemaccording to claim 17 or 18, characterized in that the unit (10) alsoincludes a counter-oscillator (29) which—to absorb oscillations of theunit—is movable counter to the linear drive (11) in the direction of theY axis.
 20. The system according to one or more of the preceding claims,characterized in that the aperture plate (18) is implemented asreplaceable for various aperture sizes.
 21. The system according to oneor more of preceding claims 10 to 20, characterized in that the firstspectral filter (19) is implemented as a filter slider having at leasttwo different filters which may be automatically replaced with oneanother and this filter slider is implemented so it may be manuallyreplaced by another filter slider.
 22. The system according to one ormore of preceding claims 10 to 21, characterized in that the beamsplitter element (26) is implemented as a carriage slider havingreplaceable mirrors.
 23. The system according to one or more of thepreceding claims, characterized in that it includes a computer ormicroprocessor for recording and processing the measurement signalsdetected by detectors (20, 20′) and for outputting data corresponding tothese signals.
 24. The system according to claim 23, characterized inthat the computer or microprocessor is additionally implemented forcontrolling the movements of the table (13), the table (13) beingimplemented as displaceable in the directions of the X and/or Y and/or Zaxes and tiltable around the X and/or Y axes.
 25. A method of excitingand measuring fluorescence on or in samples treated using fluorescentpigments using an optical system, particularly according to one ofclaims 1 through 24, in which at least: a first laser (1), which—toexcite these fluorescent pigments—emits light of a first wavelength (2),so that these fluorescent pigments emit light of a second wavelength(3); a mirror (4), for deflecting the light of the first wavelength (2),coming out of the first laser (1) and incident on the mirror (4)parallel to an optical axis (5), in the direction of a sample (6); adeflection element (7) for deflecting the light (2) from the first laser(1) onto this mirror (4); an optic (8), for producing a first focalpoint (9) for the light of the first wavelength (2), which is incidentfrom the first laser (1) and is deflected by the mirror (4); a unit(10), which includes the mirror (4) and the optic (8), mirror and opticbeing positioned fixed in relation to one another in the unit and thisunit (10) being positioned so it is movable back and forth along theoptical axis (5) and being mechanically connected to an oscillatinglinear drive (11), the optic (8) additionally being implemented as acollimator for the light of the second wavelength (3) emitted by thefluorescent pigments and the mirror (4) additionally being implementedfor deflecting this collimated light (12) diametrically opposite to thedirection of incidence of the light of the first wavelength (2) andparallel to the optical axis (5); a table (13), movable at least in thedirection of the X and Z spatial axes, for receiving sample holders (14)for samples (6) treated using at least one first fluorescent pigment,the X axis running at least approximately parallel to the surface (15)of the sample holder (14), which—for aligning the sample (6) in relationto the first focal point (9)—is movable using the table (13) in thedirection of the Z axis; an optical arrangement (16) for imaging asecond focal point (17) using the light of the second wavelength (3),which is emitted by the fluorescent pigments, collimated by the optic(8), and deflected by the mirror (4); an aperture plate (18), positionedin the second focal point (17), for masking light of the secondwavelength (3), which is incident on this aperture plate (18) at adistance from the focal point (17) greater than a specific distance; afirst spectral filter (19) for selecting a component of the light of thesecond wavelength (3) passing through the aperture plate (18); and afirst detector (20) for measuring the intensity of the light of thesecond wavelength (3) transmitted by the aperture plate (18) andselected by the first spectral filter (19); are used and which ischaracterized in that an optical system having a continuous geometricalaxis (G), which does not run through the sample, is provided, on whichthe optical arrangement (16), the second focal point (17), the apertureplate (18), the first spectral filter (19), and the first detector(20)—together with the mirror (4) and the deflection element (7)—arepositioned, this geometrical axis (G) being at least partially identicalto the optical axis (5) in the region between the mirror (4) and thefirst detector (20).
 26. The method according to claim 25, characterizedin that using the deflection element (7), which includes a highlyreflective region (21) intended for the laser beam (2) and isessentially transparent to the light of both the first wavelength (2)and the second wavelength (3) in its remaining regions, light (2) fromthe first laser (1) is deflected onto this mirror (4) in a directionparallel to the optical axis (5).
 27. The method according to claim 26,characterized in that the highly reflective region (21) of thedeflection element (7) is positioned at a distance (A) to the opticalaxis (5), which is identical to the geometrical axis (G), so that thebeam path for the light of the first wavelength (2) coming out of thelaser (1) runs parallel and at the distance (A) to these axes (5, G).28. The method according to one or more of claims 25 through 27,characterized in that—to define an optimum Z position of the movabletable (13) and/or a sample (6)—an opaque screen (22), positioned betweenthe deflection element (7) and the optical arrangement (16), is pulledout of the beam path and in which—on the basis of a series ofmeasurement signals generated during a Z movement of the table (13) bythe first detector (20) and recorded in a computer or microprocessor—theZ position of the table corresponding to the maximum of thesemeasurement signals is calculated and the table (13) is moved into thisZ position.
 29. The method according to claim 25 or 26, characterized inthat—to define an optimum Z position of the movable table (13) and/or asample (6)—an opaque screen (22), positioned between the deflectionelement (7) and the optical arrangement (16), is pushed into the beampath and in which—on the basis of a series of measurement signalsgenerated during a Z movement of the table (13) by the first detector(20) and recorded in a computer or microprocessor—the Z position of thetable corresponding to the maximum of these measurement signals iscalculated and the table (13) is moved into this Z position.
 30. Themethod according to claim 28 or 29, characterized in that the geometriccenter between the points of inflection of the rising and falling slopesof the measurement signal is taken to fix the maximum of thesemeasurement signals.
 31. The method according to one of claims 28through 30, characterized in that this method is performed at at leastthree points of a sample holder and the table (13)—in accordance withthe defined maxima for these three points—is displaced as far asnecessary in the directions of the X, Y, and Z axes and tilted aroundthe X and Y axes.
 32. The method according to claim 31, characterized inthat it is performed using a computer or microprocessor, which detectsand processes the measurement signals generated by detectors (20, 20′),outputs data corresponding to these signals, and additionally controlsthe movements of the table (13).
 33. The method according to one or moreof claims 25 through 32, characterized in that the table (13)—before themeasurement of the fluorescence on and/or in at least one sample (6)—isdisplaced in the direction of the X and/or Y axes and is stopped for theexcitation and measurement of the fluorescence—while simultaneouslyfixing the unit (10).
 34. The method according to one or more of claims25 through 32, characterized in that the table (13)—during theexcitation and measurement of the fluorescence on and/or in at least onesample (6) and while simultaneously fixing the unit (10)—is displaced inthe direction of the X and/or Y axes.
 35. The method according to one ormore of claims 25 through 32, characterized in that—for exciting andmeasuring the fluorescence on and/or in at least one sample (6)—whilesimultaneously oscillating the unit (10) in the direction of the Yaxis—the table (13) is fixed.
 36. The method according to one or more ofclaims 25 through 32, characterized in that—for exciting and measuringthe fluorescence on and/or in at least one sample (6)—whilesimultaneously oscillating the unit (10) in the direction of the Yaxis—the table (13) is displaced linearly in the direction of the Xaxis.
 37. The method according to one of claims 33 through 36,characterized in that the corresponding detector signals are recordedand processed in the computer or microprocessor.
 38. The methodaccording to one of claims 33 through 37, characterized in that theopaque screen (22) is pushed into the beam path and/or removedtherefrom.
 39. The method according to one or more of claims 33 through38, characterized in that the excitation and measurement of thefluorescence on and/or in a sample (6) is performed at a Z position ofthe movable table (13), sample holder (14), and/or a sample (6)optimized according to claims 27 through 31 and the correspondingdetector signals are recorded and processed in the computer ormicroprocessor.
 40. The method according to one or more of claims 25through 39, characterized in that only one light flash and/or a fewdiscrete light flashes per sample (6) are emitted to excite thefluorescence.
 41. The method according to one or more of claims 25through 40, characterized in that multiple lasers are used forgenerating excitation light of different wavelengths in or on multiplefluorescent pigments and multiple detectors are used for measuring thecorresponding signals.
 42. The method according to one or more of claims25 through 41, characterized in that signals of at least two lasers,fluorescent pigments, and detectors are recorded, processed, and outputsuperimposed.